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MicroRNA‑21 regulates the expression of BTG2 in HepG2 liver cancer cells.

Mao B, Xiao H, Zhang Z, Wang D, Wang G - Mol Med Rep (2015)

Bottom Line: The expression levels of miR‑21 in the HepG2 cells were significantly higher, compared with those in L02 normal liver cells.These results indicated that miR‑21 regulates cell proliferation, invasion, migration and apoptosis in HepG2 cells, which may be associated with its effects on the expression of BTG2.The results of the present study may provide a basis for targeting the miR‑21/BTG2 interaction for the treatment of HCC.

View Article: PubMed Central - PubMed

Affiliation: Cancer Center, Institute of Surgical Research, Daping Hospital, Third Military Medical University, Chongqing 400042, P.R. China.

ABSTRACT
B‑cell translocation gene 2 (BTG2) is a tumor suppressor gene, which belongs to the anti‑proliferation gene family. Our previous study demonstrated that microRNA (miR)‑21 and the expression of BTG2 were negatively correlated during hepatocarcinogenesis. The aim of the present study was to investigate the effects of miR‑21 on the growth and progression of liver cancer cells, and to determine the underlying mechanism. A luciferase reporter assay was used to demonstrate that the BTG2 gene was a direct target of miR‑21. In addition, the effects of miR‑21 on cell growth and gene expression in HepG2 human hepatocellular carcinoma (HCC) cells were analyzed using reverse transcription‑quantitative polymerase chain reaction, western blotting, an MTT assay, flow cytometry, a Transwell invasion assay and a wound healing assay. The expression levels of miR‑21 in the HepG2 cells were significantly higher, compared with those in L02 normal liver cells. The expression levels of BTG2 in liver cancer cell lines (HepG2 and Huh7) were significantly lower, compared with that in the L02 cells. These results suggested that BTG2 was the direct target gene of miR‑21. The protein expression levels of BTG2 were inhibited by high expression levels of miR‑21, and increased by inhibition of the expression of miR‑21 in the HepG2 cells. Inhibition of miR‑21 reduced cell proliferation and invasion, and increased the rate of apoptosis in the HepG2 cells. These results indicated that miR‑21 regulates cell proliferation, invasion, migration and apoptosis in HepG2 cells, which may be associated with its effects on the expression of BTG2. The results of the present study may provide a basis for targeting the miR‑21/BTG2 interaction for the treatment of HCC.

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BTG2 is a direct target gene of miR-21. (A) Protein expression levels of BTG2 were increased in HepG2 liver cancer cells transfected with the miR-21 inhibitor, compared with those transfected with the miR-21 inhibitor NC. The protein expression levels of BTG2 decreased in the HepG2 cells transfected with miR-21 mimics, compared with those transfected with the NC. (B) Position of the 3′UTR of BTG2 binding with miR-21. (C) Luciferase activity was significantly decreased in the HepG2 cells following co-ntransfection with the pYr-MirTarget-BTG2-3′U plasmid and miR-21 mimics, but was increased following co-transfection with the pYr-MirTarget-BTG2-3′U plasmid and miR-21 inhibitor, compared with the NC group. (D) Following deletion of the predicted binding site of BTG2 3′UTR, the HepG2 cells were co-transfected with miR-21 mimics and a pYr-MirTarget plasmid (vector), pYr-MirTarget-BTG2-3U plasmid (wild type) or pYr-MirTarget-BTG2-3U-Delete site plasmid (mutation). The results revealed that the luciferase activities were significantly enhanced following deletion of the predicted binding site, compared with the wild-type (mimic) group. *P<0.05, **P<0.01, the mimics NC group compared with miR-21 mimics, and the inhibitor group compared with miR-21 inhibitor group. BTG2, B-cell translocation gene 2; miR-21, microRNA-21; NC, negative control; UTR, untranslated region.
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f7-mmr-12-04-4917: BTG2 is a direct target gene of miR-21. (A) Protein expression levels of BTG2 were increased in HepG2 liver cancer cells transfected with the miR-21 inhibitor, compared with those transfected with the miR-21 inhibitor NC. The protein expression levels of BTG2 decreased in the HepG2 cells transfected with miR-21 mimics, compared with those transfected with the NC. (B) Position of the 3′UTR of BTG2 binding with miR-21. (C) Luciferase activity was significantly decreased in the HepG2 cells following co-ntransfection with the pYr-MirTarget-BTG2-3′U plasmid and miR-21 mimics, but was increased following co-transfection with the pYr-MirTarget-BTG2-3′U plasmid and miR-21 inhibitor, compared with the NC group. (D) Following deletion of the predicted binding site of BTG2 3′UTR, the HepG2 cells were co-transfected with miR-21 mimics and a pYr-MirTarget plasmid (vector), pYr-MirTarget-BTG2-3U plasmid (wild type) or pYr-MirTarget-BTG2-3U-Delete site plasmid (mutation). The results revealed that the luciferase activities were significantly enhanced following deletion of the predicted binding site, compared with the wild-type (mimic) group. *P<0.05, **P<0.01, the mimics NC group compared with miR-21 mimics, and the inhibitor group compared with miR-21 inhibitor group. BTG2, B-cell translocation gene 2; miR-21, microRNA-21; NC, negative control; UTR, untranslated region.

Mentions: According to publicly available databases, including TargetScan and PicTar (36,37), the BTG2 gene is a predicted target gene of miR-21. As shown in Fig. 7A, inhibition of the expression of miR-21 promoted the protein expression of BTG2 in HepG2 cells, compared with the NC-transfected cells. In the luciferase reporter assay, the signal in the HepG2 cells, which were co-transfected with the BTG2-3′-UTR plasmid and miR-21 mimic was significantly decreased, whereas the luciferase signal was increased in the cells co-transfected with the BTG2-3′-UTR plasmid and miR-21 inhibitor (Fig. 7B and C). Following deletion of the predicted binding site of the BTG2 3′UTR, theHepG2 cells were co-transfected with miR-21 mimics and pYr-MirTarget plasmid, pYr-MirTarget-BTG2-3U plasmid, or pYr-MirTarget-BTG2-3U-Delete site plasmid. As shown in Fig. 7D, the luciferase activities were significantly enhanced following deletion of the predicted binding site, compared with the wild-type group, suggesting that BTG2 is a direct target gene of miR-21.


MicroRNA‑21 regulates the expression of BTG2 in HepG2 liver cancer cells.

Mao B, Xiao H, Zhang Z, Wang D, Wang G - Mol Med Rep (2015)

BTG2 is a direct target gene of miR-21. (A) Protein expression levels of BTG2 were increased in HepG2 liver cancer cells transfected with the miR-21 inhibitor, compared with those transfected with the miR-21 inhibitor NC. The protein expression levels of BTG2 decreased in the HepG2 cells transfected with miR-21 mimics, compared with those transfected with the NC. (B) Position of the 3′UTR of BTG2 binding with miR-21. (C) Luciferase activity was significantly decreased in the HepG2 cells following co-ntransfection with the pYr-MirTarget-BTG2-3′U plasmid and miR-21 mimics, but was increased following co-transfection with the pYr-MirTarget-BTG2-3′U plasmid and miR-21 inhibitor, compared with the NC group. (D) Following deletion of the predicted binding site of BTG2 3′UTR, the HepG2 cells were co-transfected with miR-21 mimics and a pYr-MirTarget plasmid (vector), pYr-MirTarget-BTG2-3U plasmid (wild type) or pYr-MirTarget-BTG2-3U-Delete site plasmid (mutation). The results revealed that the luciferase activities were significantly enhanced following deletion of the predicted binding site, compared with the wild-type (mimic) group. *P<0.05, **P<0.01, the mimics NC group compared with miR-21 mimics, and the inhibitor group compared with miR-21 inhibitor group. BTG2, B-cell translocation gene 2; miR-21, microRNA-21; NC, negative control; UTR, untranslated region.
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f7-mmr-12-04-4917: BTG2 is a direct target gene of miR-21. (A) Protein expression levels of BTG2 were increased in HepG2 liver cancer cells transfected with the miR-21 inhibitor, compared with those transfected with the miR-21 inhibitor NC. The protein expression levels of BTG2 decreased in the HepG2 cells transfected with miR-21 mimics, compared with those transfected with the NC. (B) Position of the 3′UTR of BTG2 binding with miR-21. (C) Luciferase activity was significantly decreased in the HepG2 cells following co-ntransfection with the pYr-MirTarget-BTG2-3′U plasmid and miR-21 mimics, but was increased following co-transfection with the pYr-MirTarget-BTG2-3′U plasmid and miR-21 inhibitor, compared with the NC group. (D) Following deletion of the predicted binding site of BTG2 3′UTR, the HepG2 cells were co-transfected with miR-21 mimics and a pYr-MirTarget plasmid (vector), pYr-MirTarget-BTG2-3U plasmid (wild type) or pYr-MirTarget-BTG2-3U-Delete site plasmid (mutation). The results revealed that the luciferase activities were significantly enhanced following deletion of the predicted binding site, compared with the wild-type (mimic) group. *P<0.05, **P<0.01, the mimics NC group compared with miR-21 mimics, and the inhibitor group compared with miR-21 inhibitor group. BTG2, B-cell translocation gene 2; miR-21, microRNA-21; NC, negative control; UTR, untranslated region.
Mentions: According to publicly available databases, including TargetScan and PicTar (36,37), the BTG2 gene is a predicted target gene of miR-21. As shown in Fig. 7A, inhibition of the expression of miR-21 promoted the protein expression of BTG2 in HepG2 cells, compared with the NC-transfected cells. In the luciferase reporter assay, the signal in the HepG2 cells, which were co-transfected with the BTG2-3′-UTR plasmid and miR-21 mimic was significantly decreased, whereas the luciferase signal was increased in the cells co-transfected with the BTG2-3′-UTR plasmid and miR-21 inhibitor (Fig. 7B and C). Following deletion of the predicted binding site of the BTG2 3′UTR, theHepG2 cells were co-transfected with miR-21 mimics and pYr-MirTarget plasmid, pYr-MirTarget-BTG2-3U plasmid, or pYr-MirTarget-BTG2-3U-Delete site plasmid. As shown in Fig. 7D, the luciferase activities were significantly enhanced following deletion of the predicted binding site, compared with the wild-type group, suggesting that BTG2 is a direct target gene of miR-21.

Bottom Line: The expression levels of miR‑21 in the HepG2 cells were significantly higher, compared with those in L02 normal liver cells.These results indicated that miR‑21 regulates cell proliferation, invasion, migration and apoptosis in HepG2 cells, which may be associated with its effects on the expression of BTG2.The results of the present study may provide a basis for targeting the miR‑21/BTG2 interaction for the treatment of HCC.

View Article: PubMed Central - PubMed

Affiliation: Cancer Center, Institute of Surgical Research, Daping Hospital, Third Military Medical University, Chongqing 400042, P.R. China.

ABSTRACT
B‑cell translocation gene 2 (BTG2) is a tumor suppressor gene, which belongs to the anti‑proliferation gene family. Our previous study demonstrated that microRNA (miR)‑21 and the expression of BTG2 were negatively correlated during hepatocarcinogenesis. The aim of the present study was to investigate the effects of miR‑21 on the growth and progression of liver cancer cells, and to determine the underlying mechanism. A luciferase reporter assay was used to demonstrate that the BTG2 gene was a direct target of miR‑21. In addition, the effects of miR‑21 on cell growth and gene expression in HepG2 human hepatocellular carcinoma (HCC) cells were analyzed using reverse transcription‑quantitative polymerase chain reaction, western blotting, an MTT assay, flow cytometry, a Transwell invasion assay and a wound healing assay. The expression levels of miR‑21 in the HepG2 cells were significantly higher, compared with those in L02 normal liver cells. The expression levels of BTG2 in liver cancer cell lines (HepG2 and Huh7) were significantly lower, compared with that in the L02 cells. These results suggested that BTG2 was the direct target gene of miR‑21. The protein expression levels of BTG2 were inhibited by high expression levels of miR‑21, and increased by inhibition of the expression of miR‑21 in the HepG2 cells. Inhibition of miR‑21 reduced cell proliferation and invasion, and increased the rate of apoptosis in the HepG2 cells. These results indicated that miR‑21 regulates cell proliferation, invasion, migration and apoptosis in HepG2 cells, which may be associated with its effects on the expression of BTG2. The results of the present study may provide a basis for targeting the miR‑21/BTG2 interaction for the treatment of HCC.

Show MeSH
Related in: MedlinePlus